This project aims to identify the fundamental metabolic differences between cochlear inner and outer hair cells (IHC, OHC) and to determine how these differences mediate cellular responses to ototoxic agents. Reactive oxygen species (ROS), normal byproducts of metabolism, can rise to lethal levels when mitochondrial metabolism is perturbed. Numerous studies have shown that the production and destructive actions of ROS are common features of multiple hearing loss (HL) pathologies, including aminoglycoside (AG)-induced ototoxicity, noise-induced HL (NIHL), and age-related HL (ARHL). Although there is little doubt that inner ear ROS cause HL, the exact mechanism(s) responsible for ROS production is/are controversial. Recent advances in two-photon confocal imaging of endogenous NADH (metabolic intermediate) allowed the first imaging of real-time changes in mitochondrial metabolism in live cochlear preparations. Using gentamicin (GM) as a representative AG/ototoxic agent, the immediate metabolic responses (NADH fluorescence change) and subsequent production of ROS in IHCs and OHCs will be examined. This proposal pinpoints how ROS production results from a direct GM-induced inhibition of mitochondrial metabolism. In a series of experiments that specifically block mitochondrial ROS production during GM exposure, this proposal provides the first assessment of the previously proposed role of ROS-producing bioactive iron-AG complexes in mediating AG-induced ROS production in cochlear HCs. The innovative model proposed herein describes how differential IHC and OHC ROS production can occur as a consequence of a rapid GM-induced OHC-specific inhibition of NADH production, succinate dehydrogenase activity, and electron flow through the electron transport chain while IHC metabolism remains relatively unfettered. Each Specific Aim will test the validity of the proposed model of GM-induced ototoxicity due to direct modulations of mitochondrial metabolism. Results from these studies will revolutionize our knowledge of AG ototoxicity by 1) differentiating between immediate and long-term effects, 2) determining whether GM-induced ROS production results from mitochondrial dysfunction rather than bioactive iron-AG complexes, and 3) determining whether fundamental differences in IHC and OHC mitochondrial metabolism dictate differential responses to a host of ototoxic agents. By determining the exact site(s) of ROS production, this project spurs the production of new, optimized HL prevention and treatment strategies that specifically target key ROS production site(s). Agents that site- specifically reduce metabolic ROS production and subsequent HC loss will be identified as key pharmaceutical intermediates poised to reduce the ototoxicity experienced during clinical GM treatment.